In this study, we identified and characterized two murine cDNAs encoding medium-chain acyl-CoA synthetase (MACS). One, designated MACS1, is a novel protein and the other the product of the Sa gene (Sa protein), which is preferentially expressed in spontaneously hypertensive rats. Based on the murine MACS1 sequence, we also identified the location and organization of the human MACS1 gene, showing that the human MACS1 and Sa genes are located in the opposite transcriptional direction within a 150-kilobase region on chromosome 16p13.1. Murine MACS1 and Sa protein were overexpressed in COS cells, purified to homogeneity, and characterized. Among C4-C16 fatty acids, MACS1 preferentially utilizes octanoate, whereas isobutyrate is the most preferred fatty acid among C2-C6 fatty acids for Sa protein. Like Sa gene transcript, MACS1 mRNA was detected mainly in the liver and kidney. Subcellular fractionation revealed that both MACS1 and Sa protein are localized in the mitochondrial matrix. 14C-Fatty acid incorporation studies indicated that acyl-CoAs produced by MACS1 and Sa protein are utilized mainly for oxidation.